Studies with the gram-negative bacterium Escherichia coli have indicated that proteins are exported to the periplasmic space and the outer membrane of this organism by a mechanism similar to that proposed for the initial steps of protein secretion in eukaryotic cells. Using E. coli as a model, we wish to understand the various factors that determine protein localization in this organism. We have a selection procedure that enables us to isolate mutants defective in the secretion of the maltose binding protein (MBP) into the periplasm. In these mutants, a percentage (in some cases, nearly 100%) of the MBP accumulates in the cytoplasm in its unprocessed precursor form. Beginning with these mutants which harbor either point mutations or deletions in the malE gene encoding the MBP, we can obtain revertants which restore, with varying efficiency, MBP export to the periplasm. Some of the reversion mutations map at malE, others are unlinked to the malE gene and presumably alter the cell's export machinery. Thus, using these kinds of genetic approaches, we propose to define those factors that determine protein localization in E. coli. In addition to genetic analyses, we are undertaking a detailed kinetic analysis of the synthesis, export and turnover of both the wild-type MBP and mutant MBP. We are also attempting to determine if alterations in the MBP adversely affecting secretion of this protein will likewise affect the site of synthesis of this protein which normally takes place on ribosomes bound to the cytoplasmic membrane. Eventually, we hope to develop a totally in vitro system with which to study MBP synthesis, translocation and processing. Our research goals, if achieved, should provide us valuable insight into understanding the early steps in the localization process for exported proteins in both prokaryotic and eukaryotic systems.